Readers’ eye movements were recorded to examine the role of character positional frequency on Chinese lexical acquisition during reading and its possible modulation by word spacing. In Experiment 1, three types of pseudowords were constructed based on each character’s positional frequency, providing congruent, incongruent, and no positional word segmentation information. Each pseudoword was embedded into two sets of sentences, for the learning and the test phases. In the learning phase, half the participants read sentences in word-spaced format, and half in unspaced format. In the test phase, all participants read sentences in unspaced format. The results showed an inhibitory effect of character positional frequency upon the efficiency of word learning when processing incongruent pseudowords both in the learning and test phase, and also showed facilitatory effect of word spacing in the learning phase, but not at test. Most importantly, these two characteristics exerted independent influences on word segmentation. In Experiment 2, three analogous types of pseudowords were created whilst controlling for orthographic neighborhood size. The results of the two experiments were consistent, except that the effect of character positional frequency was absent in the test phase in Experiment 2. We argue that the positional frequency of a word’s constituent characters may influence the character-to-word assignment in a process that likely incorporates both lexical segmentation and identification.